Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging

Abstract

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass.

Figure 1

Scheme of the line-scan confocal microscope. (a) An optical design of the line-scan confocal microscope. The new microscope was built by modifying a commercial inverted microscope (IX71, Olympus). For versatility of the microscope, three lasers—473-nm (Blues TM50, Cobalt), 532-nm (Compass215M, Coherent), and 640-nm (Cube640-100C, Coherent)—were used as light sources. CL1, a cylindrical lens (f = 40 mm, LJ1125L1-A, Thorlabs, Newton, NJ); CL2, a cylindrical lens (f = 50 mm, LJ1695L2-A, Thorlabs); CL3, a cylindrical lens (f = 250 mm, LJ1267L1-A, Thorlabs); L1, a spherical lens (f = 250 mm, LA1461-A, Thorlabs); L2, a spherical achromatic lens (f = 250 mm, LAO-250.0-50.0/075, Melles-Griot); L3, a spherical achromatic lens (f = 300 mm, LAO-300.0-50.0/075, Melles-Griot); L4 and L5, spherical achromatic lenses (f = 100 mm, LAO-100.0-50.0/075, Melles-Griot, Albuquerque, NM); L6, a spherical achromatic lens (LAO-350.0-40.0/Specialty HEBBAR coating for 415nm to 700nm, Melles Griot); PM, a polychroic mirror (z473/532/633rpc-xt, Chroma, Muenster, Germany); GM1 and GM2, galvanometric mirrors (VM1000+, General Scanning); OL, an objective lens (UPLSAPO100XO, or UPLSAPO60XW, Olympus); S, a slit (S30R or S40R, Thorlabs). (b) Side view of the sample plane. The illumination beam (green) is focused in a line shape on the sample plane. Only the fluorescence signal near the focal plane (yellow) can filter through a confocal slit and thus be detected. (c) Scheme of 2-D image generation. The illumination line on the sample plane (green) and the fluorescence image on a CCD camera (red) are simultaneously scanned once per filming cycle of the camera.

Figure 2

Characterization of the line-scan confocal microscope. (a) Comparison of the optical-sectioning capabilities of epifluorescence microscopy (31.7 μm), HILO (6.7 μm), and the line-scan confocal microscopy (1.0 μm). (b) A line-scan confocal image of Cy3-labeled single-stranded DNA on a coverslip in the presence of 10 nM free Cy3. To make the images, 10 frames with 100-ms exposure time were averaged and background fluorescence was subtracted. Scale bar, 3μm. (c) Same as in (b), except that the image was acquired in a HILO microscope. (d) Observation of Holliday junction dynamics via line-scan confocal microscopy. (left) A model of two-state conformational dynamics of the Holliday junction. For FRET measurements, donor (green circle) and acceptor (red circle) dye labels were attached to the ends of the Holliday junction. (right) Representative fluorescence intensity (green for donor and red for acceptor) and corresponding FRET-efficiency (black) time traces. Exposure time, 100 ms. (e) FRET histogram generated from 44 molecules. The oil-immersion objective was used for all experiments.

Figure 3

Single-molecule imaging in live cells. TMR-labeled cAMP receptors of Dictyostelium discoideum were imaged on both the basal (a) and apical surfaces (b). Scale bars, 3 μm. (c) Diffusion coefficients of cAMP receptors at the apical and basal surfaces of the cell. Molecules surviving for >15 frames (54 molecules/cell for the basal surface and 30 molecules/cell for the apical surface on average) were used for the analysis. Molecules from the same amoeba cell were used to get a single diffusion-coefficient value for either the basal surface or the apical surface. The data presented are the averages of 13 cells for the basal surface, and 11 cells for the apical surface. The oil-immersion objective was used for the experiments, and the exposure time was 50 ms.

Figure 4

Superresolution fluorescence imaging. (a) Microtubules at the bottom of COS-7 were imaged via dSTORM. (Inset) Comparison between dSTORM (left) and conventional fluorescence microscopy (right). (b) Microtubule width in the dSTORM image. The cross-section profile of the microtubule (right) was generated by collecting a number of localization spots along the line perpendicular to the length of the microtubule in the white box in the dSTORM image. The histogram was well fitted to a Gaussian function with FWHM of 59 nm (right, blue lines). (c–e) Superresolution images at depths of 4 μm (c), 7.5 μm (d), and 85 μm (e), respectively. The image in e was obtained from a cell fixed on the glass slide side of the channel using the water-immersion objective. For all other experiments, the oil-immersion lens was used. Exposure time, 100 ms.